1.1 Field of the Invention
The present invention relates to microscopy and, in particular, to apparatus, methods, and systems for simultaneous atomic force microscopy and fluorescence measurements (sometimes referred to as “AFM-FM”), including but not limited to single molecules or other nano-scale structures.
We describe a combined single molecule AFM-confocal fluorescence microscope which is capable of simultaneously measuring fluorescence time traces, spectra and forces of single molecules (sometimes referred to as “smAFM-FM”).
1.2 Related Art
Atomic Force Microscopy (AFM) is a well-known type of microscopy. See, for example, U.S. Pat. No. 4,724,318 to inventor Binnig, incorporated by reference herein, for discussion of general principles of AFM. The AFM cantilever typically has a probe end on the order of nanometers radius of curvature. AFM can be used for mapping topography of very small surfaces or objects; a type of “imaging” of the surface. But also it can be used in a manner some call force spectroscopy—direct measurement of tip-sample interaction forces. Measurements have been used, for example, to measure nanoscale contacts, atomic bonding, Van der Waals forces, Casimir forces, dissolution forces in liquid and single molecule stretching and rupture forces. Thus, AFM can be used to image and manipulate molecules or nano-scale particles or structures on a variety of surfaces. This includes a three-dimensional surface profile.
The manner in which AFM tip scanning can be accomplished is either with an xyz piezoelectric scanner on which the tip can be mounted or an xyz piezoelectric scanner on which can be placed the sample to be interrogated. Very small movement of tip versus sample is actuated by control of the piezoelectric material. See, for example, U.S. Pat. No. 6,127,682 to inventor Nakamota, incorporated by reference herein, for discussion of the principles and apparatus for scanning techniques useful for AFM.
AFM is not without limitations. There can be limitations on depth of field but also total area of image. Scanning speed is also a limitation relative to other micro- and nano-imaging methods. Additional limitations are well known in the art. See, for example, U.S. Pat. No. 5,874,668 to inventors Xu and Arnsdorf, which is incorporated by reference herein, for a discussion of issues with AFM, including for biological samples.
Therefore, there is room for improvement in this technological field. For example, it could be beneficial to gather or sense other information about a sample when using AFM. There remain various needs in the technical art for ways to investigate and gain understanding for very small samples and objects.
There have been suggestions of combining AFM with other information gathering techniques or other microscopy techniques. One example is Forster Resonance Energy Transfer (FRET). A discussion of FRET and other fluorescence-based microscopy techniques is set forth in U.S. Pat. No. 6,844,150 to inventors Weiss, et al., incorporated by reference herein. U.S. Pat. No. 6,806,953 to inventors Engelhardt and Hoffmann, incorporated by reference herein, provide an example of a fluorescence-based microscope and its operation.
An example of such combined techniques is described in Sivasankar, S. and Chu, S., Nanoparticle-Mediated Nonfluorescent Bonding of Microspheres to Atomic Force Microscope Cantilevers and Imaging Fluorescence from Bonded Cantilevers with Single Molecule Sensitivity, NANO LETT. 2009, Vol. 9, No. 5, 2120-2124, incorporated by reference herein. It describes an example of an AFM subsystem and a fluorescence microscopy subsystem. While this approach has certain benefits, there remains room for improvement.
Other attempts to combine AFM and fluorescence microscopy (FM) tend to be crude combinations which are unwieldy and experimental at best. They normally require a highly skilled person (Ph. D. or grad-student level) to even attempt to operate them. They are not integrated in the sense of control, measurements, or manipulation ability.
A need has therefore been identified in the technological art for improvements in this area. For example, the article of NANO LETT, 2009, Vol. 9, No. 5, 2120-2124 has discussed some of the technological hurdles. One example is the low probability of interaction between a sample on the substrate and the AFM tip. Id. pg. 2120, ¶2. The approach there is to add a larger colloidal probe to the normally sharp AFM tip to increase the area of contact between the tip and substrate. However, this has been found to have limitations and issues.